WO2022260579A2 - Fusion protein in myasthenia gravis - Google Patents
Fusion protein in myasthenia gravis Download PDFInfo
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- WO2022260579A2 WO2022260579A2 PCT/SE2022/050556 SE2022050556W WO2022260579A2 WO 2022260579 A2 WO2022260579 A2 WO 2022260579A2 SE 2022050556 W SE2022050556 W SE 2022050556W WO 2022260579 A2 WO2022260579 A2 WO 2022260579A2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
Definitions
- the present invention generally relates to fusion proteins useful in treatment of myasthenia gravis, and in particular to the production of such fusion proteins.
- Myasthenia gravis is a relatively rare autoimmune nerve-muscle disease that causes severe muscle weakness and, in many cases, a difficult life situation for the affected patient.
- the disease affects some 200 000 people in the EU and the US together and occurs in both sexes. It generally affects younger women ( ⁇ 40 years old) and older men (> 60 years old).
- Characteristic of the disease are fatigue and muscle weakness caused by an impaired transmission of nerve impulses to muscles. This is due to an immune attack directed against the acetylcholine receptor (AChR), which is the muscle relay station and the receiver of signals from the nerve. Destruction of AChR leads to defective neuromuscular conduction of electrical impulses and a subsequent muscle weakness, which can be very serious for the patient.
- AChR acetylcholine receptor
- An aspect of the invention relates to a method of producing a fusion protein between an extracellular domain of nicotine acetylcholine receptor subunit alpha 1 (nAChRal ) and a solubility enhancing peptide.
- the method comprises solubilizing inclusion bodies comprising the fusion protein in a solubilization solution having a pH less than 11 and lacking any reducing agent to form solubilized fusion proteins.
- the method also comprises loading the solubilized fusion proteins onto an ion-exchange resin and eluting the loaded solubilized fusion proteins from the ion-exchange resin using an elution solution having a pH less than 11 and comprising a reducing agent to form a fusion protein eluate.
- the method further comprising adjusting a pH of the fusion protein eluate to at least 11 and diluting the pH-adjusted fusion protein eluate in a refolding solution having a pH of no more than 9 to form a refolded monomeric form of the fusion protein.
- Another aspect of the invention relates to an isolated fusion protein between an extracellular domain of nAChRal as defined in SEQ ID NO: 3, in which amino acid residues 129 to 140 have been replaced by a solubility enhancing peptide.
- the asparagine residue 141 in SEQ ID NO: 3 is not glycosylated.
- the invention also relates to the fusion protein of the invention for use as a medicament and for use in treatment or prophylaxis of myasthenia gravis.
- the invention further defines a method for preventing, inhibiting or treating myasthenia gravis.
- the method comprises administering an effective amount of a fusion protein according to the invention to a subject in need thereof.
- the present invention is capable of producing a fusion protein useful in treatment of myasthenia gravis, and in particular capable of obtaining the fusion protein in a monomeric form that is suitable to create or restore myasthenia gravis-specific tolerance in the immune system to the patient’s own AChR proteins that the immune system incorrectly attacks in myasthenia gravis.
- Figure 1 illustrates plasmid map of the pJ411 vector including protein 081 (TOL2).
- Figure 2 illustrates analytical SEC chromatogram of T0L2 after refolding. Monomeric T0L2 eluted at 16 ml. The major peak at 20 ml was non-protein compounds.
- Figure 3 illustrates chromatogram of AEX run. Absorbance at 280 nm (solid line) and conductivity (hatched line). TOL2 eluted at 40 mS (1200 ml).
- FIG. 4 illustrates SEC chromatogram of TOL2. Monomeric TOL2 eluted at 1200 ml.
- Figure 5 illustrates analysis of final TOL2 sample.
- Figure 6 illustrates SDS-PAGE analysis of solubilization of TOL2 at different pHs.
- Figure 7 illustrates analysis of the solubilisation of TOL2 at pH 12.5 against 7 M urea at pH 8.0.
- Figure 8 illustrates non-reduced SDS-PAGE of refolded TOL2.
- Figure 9 illustrates SDS-PAGE for ion exchange fractions with reducing agent.
- Figure 10 illustrates SDS-PAGE for ion exchange fractions without reducing agent.
- Figure 11 illustrates SDS-PAGE for ion exchange fractions (M: Marker (SeeBlue Plus2 Pre-stained Protein Standard), 1 : Input, 2: Flowthrough, 3: Wash - 0% (with 100 mM cysteine), 4: Wash - 0%, 5: Wash - 7% of I EX 1 Buffer B, 6: Elution - 25% of IEX 1 Buffer B, and 7: Strip - 100% of IEX 1 Buffer B.
- M Marker (SeeBlue Plus2 Pre-stained Protein Standard)
- 1 Input
- 2 Flowthrough
- 3 Wash - 0% (with 100 mM cysteine)
- 4 Wash - 0%
- 5 Wash - 7% of I EX 1 Buffer B
- 6 Elution - 25% of IEX 1 Buffer B
- 7 Strip - 100% of IEX 1 Buffer B.
- Figure 13 illustrates intravenous administration of a1-ECD mt abrogates EAMG development in a dose and time dependent manner.
- G-l The data are compiled from 2 independent experiments.
- Figure 14 illustrates serum levels of rat AChR and a1-ECD mt antibodies following intravenous administration of a1-ECDmt.
- B The same serum samples as in (A) were assayed for a1-ECD mt antibodies.
- Figure 15 illustrates EAMG treatment efficacy of a1-ECD mt depends on dosing frequency.
- A Average EAMG scores ( ⁇ SD); the grey bar indicates the treatment period and arrows the timing of each administration. A dotted line represents rats treated with 12 c 100 mg daily administrations for reference.
- B Average percentage of body weight change ( ⁇ SD).
- CMAP decrement each symbol corresponds to one rat and the bar shows the mean value.
- Figure 16 illustrates EAMG treatment efficacy of a1-ECD mt is superior to that of two different active treatments for MG.
- A Average EAMG scores ( ⁇ SD); the grey bar indicates the treatment period. Circles correspond to a1- ECDmt, triangles correspond to methylprednisolone and inverted triangles correspond to pyridostigmine.
- B Average percentage of body weight change ( ⁇ SD).
- Figure 17 illustrates a1-ECD m t has a short plasma half-life and is distributed to the liver, kidneys, and spleen upon intravenous administration.
- 100 g 125 l-labelled a1-ECD m t (106 cpm) were injected intravenously to healthy rats or EAMG rats on day 21 or day 40 after disease induction.
- B The rats were sacrificed six hours after injection, selected organs were collected and measured.
- Figure 18 illustrates a1-ECD m t and a1-ECD m display comparable treatment efficacies.
- A Average EAMG scores ( ⁇ SD); the grey bar indicates the treatment period. Circles correspond to a1-ECD m t and triangles correspond to a1-ECD m , while the dotted line represents untreated rats for reference.
- B Average percentage of body weight change ( ⁇ SD).
- CMAP decrement each symbol corresponds to one rat and the bar shows the mean value. In (C) dotted lines represent untreated EAMG rats and healthy rats for reference
- the present invention generally relates to fusion proteins useful in treatment of myasthenia gravis, and in particular to the production of such fusion proteins.
- the present invention is based on the usage of a disease-specific fusion protein that is employed to create or restore myasthenia gravis-specific tolerance in the immune system to the patient’s own AChR proteins that the immune system incorrectly attacks in myasthenia gravis.
- the fusion protein produced according to the invention acts as a myasthenia gravis tolerogen capable of restoring tolerance to the body’s own AChR proteins that the immune system incorrectly attacks in myasthenia gravis.
- AChR is an integral membrane protein that responds to the binding of the neurotransmitter acetylcholine.
- AChRs are typically classified as nicotinic acetylcholine receptors (nAChR) that are particularly responsive to nicotine and muscarinic acetylcholine receptors (mAChR) that are particularly responsive to muscarine.
- nAChRs are found in the central and peripheral nervous system, muscle, and many other tissues of human. At the neuromuscular junction they are the primary receptor in muscle for motor nerve- muscle communication that controls muscle contraction.
- nAChR is made up of five subunits arranged symmetrically around a central pore.
- nAChR muscle-type nAChR and neuronal-type nAChR.
- Muscle-type nAChR found at the neuromuscular junctions are either in an embryonic form, composed of ai, bi, g, and d subunits in a 2:1 :1 :1 ratio ((a bigd), or the adult form composed of ai, bi, d, and e subunits in a 2:1 :1 :1 ratio ((a bide).
- muscle-type nicotine acetylcholine receptor subunit alpha 1 (nAChRal) is encoded by the CHRNA1 gene and is presented below and in SEQ ID NO: 1 :
- nAChRal corresponds to amino acids 21 to 255 of SEQ ID NO: 1 and is presented below and in SEQ ID NO: 2:
- the fusion protein of the invention is based on the extracellular domain of nAChRal presented above and in SEQ ID NO: 2.
- the fusion protein is based on amino acid residues 1-58, 84-235 in SEQ ID NO: 2 but in which twelve amino acids (marked in bold above) of a sequence motif denoted the Cys loop have been exchanged to improve solubility properties of the fusion protein and promote effective recombinant expression while retaining the native structure.
- the amino acid sequence omitting amino acid residues 59 to 83 in SEQ ID NO: 2 is presented below and in SEQ ID NO: 3:
- the fusion protein comprising the amino acid sequence Glu129 - Gln140 in SEQ ID NO: 3 replaced by the amino acid sequence Asp132 - Thr143 from L stagnalis AChBP is presented below and in SEQ ID NO: 5: SEHETRLVAK LFKDYSSW R PVEDHRQW E VTVGLQLIQL INVDEVNQIV
- a single N-terminal methionine (M) is added to the amino acid sequence in SEQ ID NO: 5 to enable bacterial expression of the fusion protein, such as in Escherichia coli.
- M N-terminal methionine
- the fusion protein has four Cys residues forming two intramolecular disulfide bonds between Cys128 - Cys142 and Cys192 - Cys193, respectively with the amino acid numbering in accordance with SEQ ID NO: 3 and 5.
- the fusion protein has a molecular weight of 24194.47 Da and a theoretical isoelectric pH of 5.33.
- the asparagine residue 141 (Asp141) in SEQ ID NO: 3 (and 5) is not glycosylated. Asp141 forms, with the following two amino acids Cys142 and Ser142, a N-X-S sequon, which otherwise may be involved in N-linked glycosylation. However, in a preferred embodiment, no such N-linked glycosylation occurs on Asp141.
- Recombinant production of the fusion protein is complicated by the formation of dimeric and higher multimeric forms of the fusion protein, including aggregates of the fusion proteins. Such dimeric and higher multimeric forms are generally not desired when using the fusion protein in treatment of myasthenia gravis. Hence, production of the fusion protein should result in the fusion protein in monomeric form.
- An aspect of the invention relates to a method of producing a fusion protein between an extracellular domain of nAChRal and a solubility enhancing peptide.
- the method comprises solubilizing inclusion bodies comprising the fusion protein in a solubilization solution having a pH of less than 11 and lacking any reducing agent to form solubilized fusion proteins.
- the method also comprises loading the solubilized fusion proteins onto an ion-exchange resin and eluting the loaded solubilized fusion proteins form the ion-exchange resin using an elution solution having a pH less than 11 and comprising a reducing agent to form a fusion protein eluate.
- the method further comprises adjusting a pH of the fusion protein eluate to at least 11.
- the method additionally comprises diluting the pH-adjusted fusion protein eluate in a refolding solution having a pH of no more than 9 to form a refolded monomeric form of the fusion protein.
- the fusion protein is effectively expressed and produced in host cells, preferably bacterial cells, such as E. coli cells, and accumulates in high quantities in intracellular inclusion bodies.
- host cells preferably bacterial cells, such as E. coli cells
- the inventors have unexpectedly discovered that a significant drop in pH is required between solubilization of the inclusion bodies and refolding of the fusion protein in order to obtain the fusion protein in refolded monomeric form.
- performing the all the method steps in substantially the same pH, i.e., without any significant drop results in accumulation of refolded fusion protein in dimeric and higher multimeric forms, including larger aggregates.
- the fusion protein comprises multiple cysteine residues, i.e., four such cysteine residues in SEQ ID NO: 5 and 6.
- a reducing agent is included in the solutions comprising proteins having such cysteine residues in order to prevent formation of undesired disulfide bonds within a protein molecule (intradisulfide bonds) and/or between different protein molecules (inter-disulfide bonds) during the production process.
- such a reducing agent is generally included in a protein containing solution prior to loading the protein onto an ion-exchange resin for the purpose of purifying the protein by ion-exchange chromatography, such as anion-exchange chromatography.
- the method also comprises expressing the fusion protein in bacterial cells comprising an expression vector comprising a nucleotide sequence encoding the fusion protein under control of a promoter.
- the bacterial cells are E. coli cells, such as the strain BL21 (DE3) Star (Invitrogen).
- An illustrative, but non-limiting, example of an expression vector that could be used according to the invention is the expression vector pJexpress 411 (Atum).
- the nucleotide sequence encoding the fusion protein is under transcriptional control of a promoter.
- the promoter is an inducible promoter to enable control and timing of transcription of the nucleotide sequence encoding the fusion protein and thereby of production of the fusion protein.
- the promoter is an isopropyl b-d-l -thiogalactopyranoside (IPTG) inducible promoter, such as an IPTG-inducible T7 promoter.
- IPTG isopropyl b-d-l -thiogalactopyranoside
- the method also comprises lysing the bacterial cells to form a lysate comprising the inclusion bodies and centrifuging the lysate to collect the inclusion bodies.
- the inclusion bodies are also washed in at least one wash step.
- the solubilization solution used according to the present invention has a basic pH, which in addition is preferably higher than the pH of the refolding solution.
- the solubilization solution has a pH selected within an interval of from 9 to 10.
- the solubilization solution has a pH selected within an interval of from 9.2 to 9.8, and more preferably within an interval of from 9.4 to 9.6.
- Currently preferred pH value of the solubilization solution is about 9.5.
- the solubilization solution comprises urea, preferably at least 5 M urea. In a preferred embodiment, the solubilization solution comprises at least 6 M urea, and more preferably at least 7 M urea. A currently preferred concentration of urea in the solubilization solution is about 8 M.
- the solubilization solution may also comprise additional components, such as ethylenediaminetetraacetic acid (EDTA) used as metal chelating agent and glycine used to prevent pH decrease in the solubilization solution and to stabilize the fusion protein.
- EDTA ethylenediaminetetraacetic acid
- a currently preferred solubilization solution is 10 mM glycine, 8 M urea, 5 mM EDTA, pH 9.5. Hence, the solubilization solution lacks any reducing agent, such as cysteine.
- the elution solution used to elute the fusion protein loaded onto the ion-exchange resin during anion- exchange chromatography has a basic pH, which in addition is preferably higher than the pH of the refolding solution.
- the elution solution has a pH selected within an interval of from 9 to 10.
- the elution solution has a pH selected within an interval of from 9.2 to
- the elution solution has a same pH as the solubilization solution.
- the elution solution comprises a reducing agent.
- reducing agents could be used according to invention including, but not limited to, cysteine, dithiothreitol (DTT) and 2-mercaptoethanol.
- DTT dithiothreitol
- 2-mercaptoethanol a currently preferred reducing agent is cysteine.
- the elution solution comprises urea, preferably at least 5 M urea.
- the solubilization solution comprises at least 6 M urea, and more preferably at least 7 M urea.
- a currently preferred concentration of urea in the solubilization solution is about 8 M.
- the elution solution may also comprise additional components, such as EDTA and glycine.
- an elution solution is 10 mM glycine, 8 M urea, 5 mM EDTA, 10 mM cysteine, pH 9.5.
- Another example of an elution solution is 10 mM glycine, 8 M urea, 5 mM EDTA, 10 mM cysteine, 1 M NaCI, pH 9.5.
- Further examples of elution solutions include mixtures of the two examples above to form an elution solution having a NaCI concentration between 0 M and 1 M.
- the elution solution comprises a reducing agent, i.e., cysteine.
- the pH of the fusion protein eluate is adjusted to be at least 11. This pH adjustment can be performed by addition of NaOH, such as 1 M NaOH, until a desired basic pH is reached.
- the pH of the fusion protein eluate is adjusted to be within an interval of from 11 to 12.
- the solubilization solution has a pH selected within an interval of from 11.2 to
- solubilization solution is about 11.5 or 11.6, including a pH within an interval of from 11.5 and 11.6.
- the refolding solution has a pH that is significantly lower than the pH of the pH-adjusted fusion protein eluate to achieve the pH drop promoting refolding of the fusion protein into monomeric form rather than multimeric forms of the fusion protein.
- the refolding solution preferably has a basic pH, i.e., a pH above 7.
- the refolding solution has a pH selected within an interval of from 8 to 9.
- the refolding solution has a pH selected within an interval of from 8.4 to 8.8, and more preferably within an interval of from 8.5 to 8.7.
- Currently preferred pH values of the refolding solution are about 8.5 or about 8.6, including a pH within an interval of from about 8.5 to about 8.6.
- the refolding of the fusion protein is preferably performed by diluting the pH-adjusted fusion protein eluate in the refolding solution.
- Experimental data have shown that correct and efficient refolding of the fusion protein is obtained when the fusion protein is present in a comparatively low concentration.
- the solubilized fusion protein is thereby diluted in the refolding solution to induce correct refolding of the fusion protein to obtain the refolded fusion protein in monomeric form.
- diluting the pH-adjusted fusion protein eluate comprises diluting the pH-adjusted fusion protein eluate in the refolding solution at a volume ratio of less than 0.1 : 1.
- the volume ratio X : Y as used herein is between the volume of the solubilized fusion protein (relative volume X) and the volume of the refolding solution (relative volume Y).
- diluting the solubilized fusion protein comprises diluting the solubilized fusion protein in the refolding solution at a volume ratio of less than 0.075 : 1 and more preferably less than 0.05 : 1 , such as about 0.025 : 1.
- one volume of the pH-adjusted fusion protein eluate is diluted in four volumes of refolding solution, such as by slowly adding the pH-adjusted fusion protein eluate to the refolding buffer over an extended period of time, such as at least 0.5 h, preferably at least 1 h, and more preferably at least 1.5 h, such as about 2 h.
- the diluted fusion protein is then preferably incubated over an extended period of time, such as at least 1 h, preferably at least 2 h, and more preferably at least 4 h, such as at least 5 h, at least 6, h, at least 7 h, at least 8 h, at least 9 h or at least 10 h or even longer, such as at least 12 h.
- an extended period of time such as at least 1 h, preferably at least 2 h, and more preferably at least 4 h, such as at least 5 h, at least 6, h, at least 7 h, at least 8 h, at least 9 h or at least 10 h or even longer, such as at least 12 h.
- the refolding solution comprises Tris-HCI to provide a buffering environment and keeping the pH of the refolding solution stable during the refolding.
- the refolding solution preferably comprises EDTA, reducing agent (cysteine), sucrose and glycerol.
- a currently preferred refolding solution is 100 mM Tris-HCI, 5 mM EDTA, 4 mM cysteine, 400 mM sucrose, and 10 % glycerol, pH 8.6.
- the method also comprises a purifying process comprising purifying the refolded monomeric form.
- the purifying process comprises purifying the refolded monomeric form of the fusion protein by ion exchange chromatography, preferably anion exchange chromatography. This purification step removes host cell impurities and other purities from the upstream culturing and cell lysing steps. In addition, the purifying process leads to a concentration of the refolded fusion protein by removal of water.
- the method comprises loading a chromatography column with an amount of the refolded monomeric form of the fusion protein that is below a maximum protein loading capacity of the chromatography column.
- the chromatography column used in the ion exchange chromatography preferably the anion exchange chromatography, is loaded below maximum protein loading capacity of the chromatography column.
- Experimental data indicates that using a subthreshold fusion protein loading reduces the risk of eluting the fusion protein in higher multimeric forms.
- the sub-threshold loading of the chromatography column contributes to obtaining the purified refolded fusion protein mainly in the monomeric form.
- the method comprises equilibrating a chromatography column with a buffer solution comprising polyethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock copolymer.
- the chromatography column used in the ion exchange chromatography preferably the anion exchange chromatography, is preferably equilibrated by polyethylene glycol)-poly(propylene glycol)-polyethylene glycol) triblock copolymers prior to loading the column with refolded protein.
- the polyethylene glycol)-poly(propylene glycol)-polyethylene glycol) triblock copolymer is preferably selected among PLURONIC® copolymers, such as PLURONIC® F127.
- the purifying process also comprises concentrating the purified refolded monomeric form of the fusion protein by tangential flow filtration (TFF). Such a TFF achieves an additional concentration of the fusion protein. In addition, TFF could be used to achieve a buffer shift of the solution, in which the fusion protein is dissolved.
- the purifying process further comprises purifying the concentrated refolded monomeric form of the fusion protein by size-exclusion chromatography (SEC). Such SEC achieves additional purification of the fusion protein by removing any impurities, such as host cell proteins, host cell nucleotide molecules and aggregates of the fusion protein.
- the purifying process comprises a second concentration of the purified refolded monomeric form of the fusion protein by tangential flow filtration following SEC.
- the purifying process may optionally comprise one or multiple filtration steps, such as following the TFF and/or the SEC, using a filter, such as a 0.22 m filter. Such an optional filtration step may also, or alternatively, be performed following solubilization of the inclusion bodies and prior to loading the solubilized fusion proteins onto the ion-exchange resin.
- the one or more optional filtration steps remove large particles and aggregates, which may otherwise interfere with the anion-exchange chromatography or SEC.
- the resulting concentrated fusion protein may be used as active pharmaceutical ingredient (API).
- the fusion protein may be diluted to obtain a desired API concentration of the monomeric form of the fusion protein.
- the diluting step is performed at a temperature of no more than 10°C. In a preferred embodiment, the diluting step is performed at a temperature selected within an interval of from 4°C to 10°C. In a preferred embodiment, the above described steps of the purifying process, or at least a portion of these steps, are preferably also conducted in a temperature of no more than 10°C.
- the present invention also relates to a fusion protein between an extracellular domain of nAChRal as defined in SEQ ID NO: 3, in which amino acid residues 129 to 140 have been replaced by a solubility enhancing peptide.
- the asparagine residue 141 in SEQ ID NO: 3 is not glycosylated.
- the fusion protein comprises the extracellular domain of nAChRal as defined in SEQ ID NO: 3, in which amino acid residues 129 to 140 have been replaced by amino acid residues 132 to 143 from L. stagnalis AChBP as defined in SEQ ID NO: 4.
- the fusion protein comprises, preferably consists of, the amino acid sequence as defined in SEQ ID NO: 5 or 6.
- the fusion protein consists of an N-terminal methionine residue followed by the extracellular domain of nAChRal as defined in SEQ ID NO: 3, in which amino acid residues 129 to 140 have been replaced by amino acid residues 132 to 143 from L stagnalis AChBP as defined in SEQ ID NO: 4.
- the fusion protein consists of the amino acid sequence as defined in SEQ ID NO: 6.
- the fusion protein is preferably in a monomeric form.
- the monomeric form of the fusion protein is preferably folded in the same way as the wild-type extracellular domain of nAChRal, and more preferably in the same way as the extracellular domain of nAChRal is folded in the adult form of the muscle-type nAChR ((a bide).
- the fusion protein has a first disulfide bond between cysteine residue 128 in SEQ ID NO: 3 and 5 and cysteine residue 142 in SEQ ID NO: 3 and 5 and a second disulfide bond between cysteine residue 192 in SEQ ID NO: 3 and 5 and cysteine residue 193 in SEQ ID NO: 3 and 5.
- the fusion protein has a molecular weight of about 24 kDa.
- An embodiment also relates to a nucleotide sequence encoding a fusion protein according to the invention.
- a nucleotide sequence also referred to as nucleic acid sequence or nucleotide or nucleic acid molecule herein, refers to a polymer composed of nucleotides, such as ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and/or synthetic non-naturally occurring analogs thereof, linked via phosphodiester bonds, related naturally occurring structural variants, and/or synthetic non-naturally occurring analogs thereof.
- nucleotide sequences are deoxyribonucleic acid (DNA) sequences, ribonucleic acid (RNA) sequences, complementary DNA (cDNA) sequences and messenger RNA (mRNA) sequences.
- nucleotide sequences are possible to encode a single fusion protein due to the degeneracy of the genetic code and a single amino acid may be coded for by more than one codon.
- An example of a nucleotide sequence encoding a fusion protein of the invention is shown in SEQ ID NO: 7.
- the embodiments are, however, not limited to this particular example of nucleotide sequences but also include variants thereof having a different nucleotide sequence than the one shown in any of SEQ ID NO: 7 but still, due to the degeneracy of the genetic code, encodes the same fusion protein as the nucleotide sequence shown in the any of SEQ ID NO: 7.
- such a different nucleotide sequence may be a codon optimized version of the nucleotide sequence.
- a codon optimized version of a nucleotide sequence refers to a nucleotide sequence where the codons have been optimized with regard to a particular cell used to express the polypeptide from the nucleotide sequence.
- tRNAs transfer RNAs
- Codon optimization of a nucleotide sequence thereby involves changing codons to match the most prevalent tRNAs, i.e., to change a codon recognized by a low prevalent tRNA with a synonymous codon recognized by a tRNA that is comparatively more prevalent in the given cell. This way the messenger RNA (mRNA) from the codon optimized nucleotide sequence will be more efficiently translated.
- the codon and the synonymous codon encode the same amino acid.
- An embodiment relates to a vector, in particular an expression vector, comprising a nucleotide sequence of the invention under transcription control of a promoter.
- promoter refers to a nucleic acid sequence which has functions to control the transcription of a polypeptide-coding nucleotide sequence (gene), and is located upstream with respect to the direction of transcription of the transcription initiation site of the polypeptide-coding nucleotide sequence.
- Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art.
- the promoter is preferably selected based on the type of host cell that will be used to produce the polypeptide encoded by the nucleic sequence included in the expression vector under transcriptional control of the promoter.
- Illustrative, but non-limiting, examples of a promoter that could be used for expression of the nucleotide sequence in a bacterial cell, such as an E. coli cell include the lac promoter, the T7 promoter, the Tac promoter, and the trp promoter.
- Illustrative, but non-limiting, examples of a promoter that could be used for expression of the nucleotide sequence in a yeast cell include the AOX1 promoter, the LAC4 promoter, the GAL1 promoter, the GAL2 promoter, the GAL7 promoter, the GAL10 promoter, the TEF1 promoter, the PGK1 promoter, the PDC1 promoter, the EN01 promoter, the TPI1 promoter, and the G3P promoter.
- a promoter that could be used for expression of the nucleotide sequence in insect cells is the polyhedrin promoter of baculovirus.
- Illustrative, but non-limiting, examples of a promoter that could be used for expression of the nucleotide sequence in a mammalian, such as human, cell include the CMV promoter, the SV40 promoter and the EF-1 promoter.
- a nucleotide sequence is under transcription control of a promoter when the promoter is located relative to the nucleotide sequence to control transcription of the nucleotide sequence into an mRNA sequence.
- the vector is preferably an expression vector, i.e., a vector comprising at least one nucleotide molecule comprising a fusion protein-coding nucleotide sequence that can be expressed, such as transcribed and translated, in a host cell comprising the expression vector.
- the expression vector is in an embodiment selected among DNA molecules, RNA molecules, plasmids, episomal plasmids and virus vectors.
- virus vectors include a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a baculovirus and a hybrid vector.
- a related embodiment defines a host cell comprising an expression vector of the invention.
- the host cell can then be used to express the fusion protein-coding nucleotide sequence in the expression vector to thereby produce a polynucleotide of the invention.
- the host cell could, for instance, be a bacterial cell, such an E. coli cell, a yeast cell, such as a S. cerevisiae cell, a P. pastoris cell, a Y. lipolytica cell or a K. lactis cell, an insect cell, such as a Lepidoptera cell, or a mammalian cell, such as a human cell.
- the fusion proteins of the invention can be used as tolerogens as they are capable of restoring tolerance to the body’s own proteins that the immune system incorrectly attacks in autoimmune disease.
- the present invention also relates to a fusion protein of the invention for use as a medicament and for use in treatment or prophylaxis of myasthenia gravis.
- a related aspect of the invention defines the use of the fusion protein for the manufacture of a medicament for treatment or prophylaxis of myasthenia gravis.
- Treatment or treating as used herein means an approach for obtaining beneficial or desired results, including clinical results.
- Beneficial or desired clinical results could include, for instance, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of myasthenia gravis, stabilized state of myasthenia gravis, i.e., prevent worsening, preventing spread of myasthenia gravis, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of myasthenia gravis, and remission.
- Treatment or treating may also prolong survival as compared to expected survival if not receiving any treatment.
- Preventing or prophylaxis as used herein means an approach in which a risk of developing myasthenia gravis is reduced or prevented, including prolonging or delaying myasthenia gravis development.
- a patient predisposed to develop a disease such as due to genetic or hereditary predisposition, could benefit for administration of the polypeptide or a pharmaceutical composition comprising the polypeptide to prevent, reduce the risk of, delaying and/or slowing development of myasthenia gravis.
- the fusion protein may be administered to a subject or patient in need thereof in the form of a pharmaceutical composition comprising the fusion protein of the invention.
- the pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable additives including, but not limited to, carriers, vehicles, diluents, adjuvant, aroma, preservatives and/or excipients.
- a pharmaceutically acceptable carrier or vehicle is an injection solution, such as saline or a buffered injection solution.
- the pharmaceutical composition may, for instance, be in the form of a tablet, a capsule, powder, nanoparticles, a solution, such as an injection solution, a transdermal patch or a suppository.
- a currently preferred pharmaceutical composition is an aqueous injection solution comprising the fusion protein at 3.0 mg/mL in 40 mM Tris, 150 mM NaCI, pH 8.5.
- the pharmaceutical composition preferable comprises an effective amount of the fusion protein.
- effective amount indicates an amount effective, at dosages and for periods of time necessary to achieve a desired result. Effective amounts may vary according to factors, such as the disease state, age, sex, weight of the patient.
- the patient is preferably a human patient.
- a further aspect of the invention relates to a method for preventing, inhibiting or treating myasthenia gravis.
- the method comprises administering an effective amount of a fusion protein or pharmaceutical composition of the invention to a subject in need thereof.
- an effective amount of a nucleotide sequence and/or expression vector of the embodiment, or a pharmaceutical composition comprising the nucleotide sequence and/or expression vector is administered to the subject in need thereof.
- the fusion protein or the pharmaceutical composition may be administered to the subject according to various routes including, for instance, intravenous, subcutaneous, intraperitoneal, intramuscular, topical, nasal, buccal, sublingual or oral administration, or administration via the respiratory tract, such as, in the form of an aerosol or an air-suspended fine powder.
- This examples describes generation of an expression system in Escherichia coli BL21 Star(DE3) under control of an inducible T7 promoter capable of expressing TOL2 (SEQ ID NO: 6) in high yields.
- the nucleotide sequence (SEQ ID NO: 7) encoding TOL2 was cloned into the expression vector pJ 411 ( Figure 1).
- E. coli BL21 Star (DE3) cells were transformed with the plasmid pJexpress 411-TOL2 by heat shock procedure following manufacturer’s protocol (Invitrogen: 30 min 4°C, 30 sec 42°C). Two different amounts of DNAs (5 and 10 ng) were used. Transformed cells were spread on LB agar plates containing kanamycin (50 Mg/mL). Five colonies were selected from the first plate and re-streaked in a new plate.
- An induction assay was performed to study protein expression.
- a volume of 0.2 mL of one vial of the precell banking of the five colonies was used to inoculate 50 mL of fresh LB medium without antibiotics.
- the culture was maintained at 37 ⁇ 1 °C and 225 ⁇ 10 rpm and O ⁇ eoo was monitored until it reached 1 ⁇ 0.2.
- 1 mL of culture was taken as a pre-induction sample (TO) and then, the culture was induced by the addition of 1 mM of IPTG.
- the 50 mL were split and half of it was maintained at 37°C and half of the material was put at 30°C in a second incubator. Four hours later, a 3 mL sample was taken as post-induction sample (T1).
- the cryopreservation medium used was the same recipe but including an extra 20% of glycerol.
- Plasmid identity In order to confirm the sequence of the DNA fragment corresponding to the coding protein region, isolated colonies from RCB in a TSA plate were sequenced.
- More than 50 colonies were obtained from each of the transformation (with 5 and 10 ng of plasmid).
- the plates chosen for the selection of the colonies were the one with 10 ng and 50 m ⁇ of material spread. Fibe colonies were fully characterized.
- the growth capacity was the same for all E. coli BL21Star(DE3)-TOL2 clones reaching maximum OD6oo of 7 after 11 h of culture.
- the expression system E. coli BI21 Star(DE3)-pJ411 TOL2 was able to grow in a rich media, such as LB medium, reaching high values of OD6oo in a few hours.
- the band of about 25 kDa also appeared in the insoluble fraction and was not present in the soluble fraction. This means that the protein was present inside the cells forming inclusion bodies.
- the percentage of the overexpressed protein in the total extract sample (TE, lanes 4 and 7) was calculated by densitometry of the bands within that lane with the software of Imagelab (Syngene). The percentage of the band for each colony at each temperature is shown in Table 2.
- SDS-PAGE result showed an overexpression of the protein with a similar Mw to the monomer of T0L2.
- a WB was performed using an anti-T0L2 antibody. A single band in the TE and IF samples was observed. The single band had a size of about 25 kDa. It was considered that the WB gave enough information about the identity of the protein of interest since a clear band of the expected Mw was observed.
- Isolated colonies were subject to plasmid identity analysis.
- the DNA sequence of the region coding for the protein of interest was 100% identical compared with the original sequence.
- the manufacture of the RCB did not affect the DNA, preserving the original sequence.
- This example describes a process of producing and purifying monomeric TOL2.
- Example 1 comprising E. coli BL21 Star(DE3)- TOL2 was thawed using the Multitron Incubator at 37°C during 5 minutes.
- the content of the thawed vial was homogenized in the biosafety cabinet and 1 mL from the vial was added to 500 ml Erlenmeyer flask. Further, 100 ml of the inoculated media was transferred to a 500 mL Erlenmeyer flask. Thereafter, the culture was incubated at 37°C and 230 rpm inside the Multitron Incubator until desired OD600 of 1.440 AU was reached after 4 h and 35 min. Inoculum Expansion
- F-100 medium 50 g/L glycerol, 0.88 kg/L base medium (2.272 g/L citric acid, 6.817 g/L KH2PO4, 11.361 g/L K2HPO4, 6.817 g/L Na 2 HP04, 4.543 g/L (NH ⁇ SC )
- base medium 2.272 g/L citric acid, 6.817 g/L KH2PO4, 11.361 g/L K2HPO4, 6.817 g/L Na 2 HP04, 4.543 g/L (NH ⁇ SC )
- the feed (120.00 g/L yeast extract, 5 g/L MgSC FhO, 5 g/L (NFU ⁇ SC , 700 g/L glycerol, 24.50 g/L simethicone 9%) was started at 13 hrs 19 min after inoculation with a flow rate of 28.2 mL/min.
- the induction phase was started 20 minutes after feed start by loading 0.202 L of 200 mM IPTG.
- the induction phase lasted 8 hours, and optical density (OD600) at the end of induction was 188.50 AU.
- the feed pump stopped, and temperature was adjusted to 5°C in order to start the cooling phase.
- the product transfer line was connected to a Westfalia centrifuge inlet.
- the centrifugation of the biomass harvest was performed using the following operating parameters: 11650 rpm centrifuge speed; 5.0% feed flow rate; 3.0 bar backpressure of supernatant line; 2.4 bar seal pressure; 0.4 min of discharge interval (5 partial discharges followed by a total discharge).
- the harvesting process lasted 1 h 30 min and 48.165 kg of biomass was collected into a storage tank.
- Re-suspension buffer 50 mM Tris-Base, 5 mM EDTA, pH 8.0
- the re-suspended biomass was stirred at speed of 150 rpm for 29 minutes.
- the temperature during the resuspension was kept at 8°C ( ⁇ 10°C).
- Inclusion bodies were collected from the lysate product by centrifugation using a CEPA Z61 centrifuge at 16000 rpm at a temperature of 4°C to form a centrifuged pellet.
- Centrifuged pellet was re-suspended with 78.040 L of first wash buffer (50 mM Tris, 5 mM EDTA, 1 M NaCI, pH 8) and stirred at speed of 146 rpm for 30 minutes. The temperature during the wash was kept at 9°C ( ⁇ 10°C). The washed inclusion bodies were centrifuged using CEPA centrifuge at 16040 rpm speed with 24 L/h feed flow during 4 hours and 35 minutes. Once the product was centrifuged, the equipment was fed with 7 L of the first wash buffer in order to drain the product from centrifugation system.
- first wash buffer 50 mM Tris, 5 mM EDTA, 1 M NaCI, pH 8
- the pellet of inclusion bodies was re-suspended with 5.020L of second wash buffer (50 mM Tris-HCI 50, 5 mM EDTA, 0.25% DOC, pH 8) and stirred at speed of 150 rpm for 28 minutes. The temperature during the wash was kept at 9°C ( ⁇ 10°C).
- the washed inclusion bodies were centrifuged using CEPA centrifuge at 16050 rpm speed with 9.5 L/h feed flow during 3 hours and 10 minutes. Once the product was centrifuged, the equipment was fed with 7 L of the second wash buffer in order to drain the product from centrifugation system.
- the pellet of inclusion bodies was re-suspended with 4.941 L of the second wash buffer (50 mM Tris-HCI, 5 mM EDTA, 0.25% DOC, pH 8) and stirred at speed of 150 rpm for 30 minutes. The temperature during the wash was kept at 9°C ( ⁇ 10°C). The washed inclusion bodies were centrifuged using CEPA centrifuge at 16000 rpm speed with 9.0 L/h feed flow during 4 hours and 27 minutes. Once the product was centrifuged, the equipment was fed with 7 L of the second wash buffer in order to drain the product from centrifugation system.
- the second wash buffer 50 mM Tris-HCI, 5 mM EDTA, 0.25% DOC, pH 8
- the pellet of inclusion bodies was re-suspended with 44.24 L of third wash bufferF (50 mM Tris-Base, 5 mM EDTA, pH 8.0) and stirred at speed of 150 rpm for 30 minutes. The temperature during the wash was kept at 9°C ( ⁇ 10°C).
- the washed inclusion bodies were centrifuged using CEPA centrifuge at 16030 rpm speed with 24.0 L/h feed flow during 3 hours and 25 minutes. The heavy phase (inclusion bodies) was collected. Once the product was centrifuged, the equipment was fed with 7 L of the third wash buffer in order to drain the product from centrifugation system. The total weight of inclusion bodies obtained from centrifugation was 3.484 kg.
- TOL2 inclusion bodies (about 7 g) were re-suspended in 250 ml solubilization buffer (10 mM Na2P04, 8 M urea, 5 mM EDTA, 100 mM cysteine, pH 11.6). The solution was stirred at room temperature and sonicated 4x2 min total time (15 s on, 15 s off, 70% amplitude) to re-suspend the inclusion bodies completely. The mixture was then stirred for 2 h at room temperature. Unsolubilized material was pelleted by centrifugation (38 000 g, 1.5 h, 4°C). The solubilization suspension sample was kept at 4°C until refolding.
- 10 L refolding buffer 100 mM Tris-HCI, 5 mM EDTA, 4 mM cysteine, 400 mM sucrose, 10 % glycerol, pH 8.6 was equilibrated at 5°C overnight and the solubilization suspension sample was added slowly over night (ca 10 h) in a continuous mode with stirring.
- Refolded TOL2 was analyzed with analytical size exclusion chromatography (SEC) (Superdex200 10/300), see Figure 2.
- SEC analytical size exclusion chromatography
- Refolded TOL2 was loaded onto a XK 50/30 packed with OSepharose FF (final bed volume 360 ml) equilibrated with Buffer A (40 mM Tris, 0.02% PLURONIC® F127, pH 8.5). The bound protein was washed with the same buffer containing 180 mM NaCI. Elution of TOL2 was made with stepwise increases of NaCI to 350 mM and 600 mM ( Figure 3).
- Solubilization buffer 10 mM glycine, 8 M urea, 5 mM EDTA, 100 mM cysteine, pH 9.5
- the column volume was 5 ml and fraction volume 1 ml. Selected fractions were analyzed on reduced SDS-PAGE.
- TOL2 inclusion bodies (1.79 g) were re-suspended in 120 ml solubilization buffer (10 mM glycine, 8 M urea, 5 mM EDTA, 100 mM cysteine, pH 9.5).
- solubilization buffer 10 mM glycine, 8 M urea, 5 mM EDTA, 100 mM cysteine, pH 9.5.
- a handheld mixer (ULTRA-TURRAX®) was used to dissolve inclusion body clumps. The mixture was stirred at room temperature for 2 h and sonicated 4 x 2 min total time (15 s on, 5 s off, 70% amplitude) while kept on ice to re-suspend the inclusion bodies completely. Unsolubilized material was pelleted by centrifugation (38,000 g, 1.5 hm 4°C). 1.408 mg/ml solubilized TOL2 was obtained at a total volume of 112 ml.
- the sample was loaded via peristaltic pump connected to the AKTA to observe the absorbance overtime while not contaminating the FPLC system.
- Solubilization buffer 10 mM glycine, 8 M urea, 5 mM EDTA, pH 9.5
- the column volume was 5 ml and fraction volume 1 ml. Selected fractions were analyzed on reduced SDS-PAGE.
- TOL2 inclusion bodies (1.18 g) were re-suspended in 100 ml solubilization buffer (10 mM glycine, 8 M urea, 5 mM EDTA, pH 9.5).
- solubilization buffer 10 mM glycine, 8 M urea, 5 mM EDTA, pH 9.5.
- a handheld mixer (ULTRA-TURRAX®) was used to dissolve inclusion body clumps. The mixture was stirred at room temperature for 2 h and sonicated 4 c 2 min total time (15 s on, 5 s off, 70% amplitude) while kept on ice to re-suspend the inclusion bodies completely. Unsolubilized material was pelleted by centrifugation (38,000 g, 1.5 hm 4°C). 1.694 mg/ml solubilized TOL2 was obtained at a total volume of 112 ml.
- the sample was loaded via peristaltic pump connected to the AKTA to observe the absorbance overtime while not contaminating the FPLC system.
- AEX buffers were prepared. 260 ml Workbeads 40Q column was washed with 2 M NaCI and Milli-Q water.
- Wash 1 Solubilization buffer + 100 mM cysteine - 2 CV AEX buffer A: 10 mM glycine, 8 M urea, 5 mM EDTA and 20 mM cysteine, pH 9.5 AEX buffer B: 10 mM glycine, 8 M urea, 5 mM EDTA, 20 mM cysteine and 1 M NaCI, pH 9.5 Wash 2: Step gradient on AKTA: 100 % AEX buffer A - 0% of AEX buffer B - 3 CV (until the UV & conductivity stabilizes)
- EXAMPLE 6 Production and purification of monomeric TOL2 This example describes a process of producing and purifying monomeric TOL2.
- TOL2 inclusion bodies were re-suspended in solubilization buffer (10 mM glycine, 8 M urea, 5 mM EDTA, pH 9.5) at 1 g TOL2 inclusion body per 30 ml solubilization buffer. The solution was homogenized for 10 cycles of 10 s and stirred for 2 hours.
- solubilization buffer 10 mM glycine, 8 M urea, 5 mM EDTA, pH 9.5
- Refolding pH-adjusted TOL2 eluate was added dropwise into a refolding buffer (0.1 M Tris-HCI, 5 mM EDTA, 5 mM cysteine, 0.4 M sucrose, 10 % glycerol, pH 8.6) to achieve a dilution ratio of 1/5 (1 volume of pH-adjusted TOL2 eluate + 4 volumes of refolding buffer) for a total addition time of 2 hours, at a temperature ⁇ 10°C.
- the refolding TOL2 was incubated 6-10 hours with magnetic stirring, at a temperature ⁇ 10°C.
- the system was equilibrated with 10 L/m 2 water for injection (WFI) and 10 L/m 2 buffer (40 mM Tris-HCI, 150 mM NaCI, pH 8.5).
- WFI water for injection
- 10 L/m 2 buffer 40 mM Tris-HCI, 150 mM NaCI, pH 8.5.
- the TOL2 eluate from AEX2 was concentrated to approximatively 2.5-3.0 g/L.
- the concentrated TOL2 was filtrated through a 0.2 m Sartopore 2 Midicap filter and the filtrate was collected.
- the SEC eluate was filtrated through a 0.2 m Sartopore 2 Midicap filter and the filtrate was collected.
- Myasthenia gravis is a CD4+ T cell-dependent antibody-mediated autoimmune disease, which leads to destruction of the skeletal muscle nicotinic acetylcholine receptor (AChR) at the neuromuscular junction resulting in the hallmark MG symptoms of muscle weakness and fatigue.
- Antibodies against the AChR are found in a majority of patients (-85%), while fewer patients have antibodies against the muscle specific kinase (-9%), the low-density lipoprotein receptor-related protein 4 (-2%), or other less common targets.
- the AChR is a Q14 transmembrane glycoprotein composed of five subunits with a stoichiometry of (a1)2b1gd in fetal or denervated muscles and (a1)2b1ed in adult muscles. Each subunit has a highly structured extracellular domain (ECD), which contains the disease-relevant autoantibody binding sites. Among the different ECDs, that of the a1 subunit (a1-ECD) is the primary antibody target in the autoimmune attack and most evidence so far suggests that the a1-ECD directed antibodies are the most pathogenic. Although MG is an antibody-mediated disease, high affinity autoantibody production by B cells is dependent on CD4+T cell activity.
- AChR-reactive CD4+T cells have been found in MG patients, while T cell recognition of the AChR has been examined extensively and several studies have identified T cell reactive auto-peptides, in particular from the a1-ECD. Taken together, both the antibody and T cell reactivities point to the a1-ECD as being a disease-specific antigen of particular interest in MG.
- MG cardiovascular disease
- cholinesterase inhibitors corticosteroids
- immunosuppressants plasmapheresis
- plasmapheresis intravenous immunoglobulin
- monoclonal antibodies or thymectomy treatments are not disease-specific and can cause significant side- effects. They can alleviate symptoms, but they are not curative. Therefore, lifelong immunosuppressive therapy is often required but some patients may prove treatment refractory.
- the ideal therapy would be disease-specific and target efficiently only the pathogenic autoreactive component of the immune system.
- Antigen-specific immune tolerization for treatment of autoimmune diseases may specifically abrogate autoimmunity without hampering normal immune function.
- Induction of tolerance via intravenous injection is also possible.
- administration of soluble or nanoparticle-carried antigens via the intravenous route has recently rendered positive results against autoimmune diseases, such as multiple sclerosis, Graves’ disease, and celiac disease, in the clinic, pointing to the intravenous route of administration as promoting a tolerogenic setting suitable for antigen-specific immune tolerance approaches.
- 6- to 7-week-old female Lewis rats (weighing 120-135 g) were obtained from the animal breeding unit of the Department of Animal Models for Biomedical Research of the Hellenic Pasteur Institute. They were maintained in the rodent unit of the Department, in plastic cages with wire mesh lids and 4 cm thick wood- shavings bedding (four rats per 1,600 cm 2 cage). Upon symptom manifestation they were provided with water gels and soft food at the bottom of the cages throughout the remaining experiment. All experiments described were approved by the Institute Ethics Board and conducted according to the regulations and guidelines for animal care (EU Directive 2010/63/EU for animal experiments).
- Synthesis of a1-ECDmt and a1-ECD m a1-ECDmt consists of the ECD of the human AChR a1 subunit, mutated by having its Cys-loop exchanged for that of the homologous acetylcholine binding protein from the snail Lymnaea stagnalis, and tagged with a Flag-tag and a 6-His-tag at its N- and C-terminal ends, respectively.
- a1-ECD m t was expressed in the yeast Pichia pastoris as a soluble secreted polypeptide and purified by means of metal- affinity chromatography followed by size exclusion chromatography as previously described ⁇ Int J Biol Macromol (2014) 63: 210-217).
- a1-ECD m (TOL2, SEQ ID NO: 6) consists of the same elements as a1-ECDmt.
- a1-ECD m was expressed in Escherichia coli strain NEB express (New England Biolabs Inc. USA) using a modified version of the pTrc99A-vector (Pharmacia AB, Sweden) harboring the knr gene and in which the gene encoding a1- ECDm was under transcriptional control by an IPTG-inducible Trp/Lac promoter.
- coli cells were cultured in terrific broth (Thermofisher Scientific, USA) at 37°C and a1-ECD m expression was induced with 1 mM IPTG at an O ⁇ eoo of about 0.6.
- a1-ECD m accumulated in inclusion bodies in high quantities.
- a1-ECD m -containing inclusion bodies were purified by cell-disruption in lysis buffer (0.1 M Tris, 5 mM EDTA, pH 8.5) followed by repeated washings in 2 M Urea, 2% Triton X-100 in lysis buffer and finally solubilized in 40 mM Tris, 8 M Urea, 5 mM EDTA, pH 8.5.
- Refolding of a1-ECD m was performed in 40 mM Tris, 50 mM NaCI, 1 M Urea, 10% Glycerol, 5% Sucrose pH 8.5 overnight at 4°C.
- Refolded a1-ECD m was further purified by anion exchange chromatography on Q Sepharose FF at pH 7.4 and size exclusion chromatography (SEC) on Superdex 200 pg.
- SEC size exclusion chromatography
- a1- ECDmpurity was >90% with endotoxin levels below ⁇ 1 EU/mg.
- the overall yield of purified a1-ECD m was about 80 mg/L of E. coli culture.
- a1-ECD m was frozen in storage buffer (30 mM NaP, 0.3 M NaCI, pH 7.4) and stored at -80°C.
- rats were anaesthetized with 2% isoflurane supplemented with oxygen. They were injected subcutaneously in both hind footpads and at three sites in the lower back with a total of 80 mg a1-ECDmt prepared as described above, or PBS for controls, in CFA (Becton, Dickinson and Company) supplemented with 2 mg/ml inactivated Mycobacterium tuberculosis H37RA (Becton, Dickinson and Company), in a final volume of 250 ml. Serum samples were collected from the rats by tail vein blood sampling at different timepoints during the experiments and used for anti-AChR antibody quantitation as described in Statistical Analysis.
- CFA Becton, Dickinson and Company
- H37RA Mycobacterium tuberculosis H37RA
- rats were treated intranasally or intravenously with a1-ECD m tor a1- ECDm starting 7, 21 or 40 days after EAMG induction.
- the amount of protein was 100 g administered in a volume of 10 mI per nostril, or 100, 500 or 1 ,000 g administered in a volume of 200mI in tail vein.
- rats were treated with 1 mg methylprednisolone (Solumedrol, Pfizer) injected IP in a volume of 100 ml or 18.5 mg/kg pyridostigmine (Mestinon, Meda Pharma GmbH), administered via oral gavage in a volume of 200 ml.
- Solumedrol, Pfizer 1 mg methylprednisolone
- pyridostigmine Mestinon, Meda Pharma GmbH
- the rats were monitored once a week for the first 4 weeks after EAMG induction and daily thereafter. Body weight was recorded and clinical score was observed on a flat bench before and after exercise and graded based on the presence of the following symptoms: tremor, hunched posture, reduced strength/mobility and dropped head. Exercise consisted of repetitive grasping and pulling of a 350 g grid while being held by the base of the tail for 30 seconds ( Muscle Nerve (1990) 13: 485-492). EAMG scores were evaluated as follows: 0: normal strength, no symptoms; 1 : normal before exercise, symptoms observed after exercise due to fatigue; 2: symptoms present without exercise; 3: severe symptoms at rest, hind limb paralysis, no grip; 4: moribund (Exp Neurol (2015) 270: 18-28). To minimize investigator bias, the animals were scored by two investigators, one of which was blinded to the treatment groups, and the average scores were used in the analyses.
- the rats were anaesthetized with 2% isoflurane.
- CMAP compound muscle action potential
- the tibialis anterior muscle was examined. A grounding electrode was placed subcutaneously at the upper back; a stimulating electrode was inserted at the base of the tail to stimulate the sciatic nerve; a recording electrode was placed in the center of the tibialis muscle and a reference electrode more distally at the tendon. A set of 10 supramaximal stimuli at 3 Hz were delivered and the CMAP recorded. The average decrement for each muscle was calculated from at least three separate readings. a1-ECDmt Distribution Studies
- 125 l -labeled a1 -ECDmt equivalent to 106 cpm was mixed with unlabeled a1 -ECDmt to a total of 100 g protein, which was injected intravenously to healthy or EAMG rats.
- Blood samples were collected from the tail artery at specific time points and organs were collected for analysis 6 hours after injection. Radioactivity was measured in a 1470 Wizard g-counter. To calculate the organ distribution, the labelling attributed to the blood content of each organ was estimated and subtracted ( Mol Pharmaceut (2014) 11:1591-1598).
- RIPA forRatAChR and ai -ECDmt Antibody Quantitation a1 -ECDmt or a-bungarotoxin (Sigma-Aldrich, USA) were labeled with 125 l using the chloramine T method. Following, the antibodies in test serum samples were quantified using RIPA. In brief, for the detection of a1 -ECDmt antibodies, 125 l-a1 -ECDmt (50,000 cpm) was incubated for 2 h at 4°C with serial dilutions of the test serum (made in normal rat serum). The total volume of rat serum used was 2 ml. Then 10 ml of rabbit anti-rat serum were added and incubated overnight at 4°C.
- rat AChR was prepared from denervated rat muscle and labeled for 1 h at 4.C with 125 l-a- bungarotoxin (50,000 cpm) before incubation with the test serum serial dilutions. All the following steps were performed as described previously for the a1-ECD m t antibodies.
- EAMG Treatment Efficacy of cd -ECDmt is Superior to that of two Different Active Controls
- a cholinesterase inhibitor pyridostigmine
- a corticosteroid methylprednisolone
- Immunotherapies commonly used to treat MG are unspecific and associated with serious long-term side effects. To mitigate this, some immunotherapies currently in development are directed against pathological mechanisms more specific to MG. These therapies, such as complement C5 inhibitors and neonatal Fc receptor inhibitors, may be associated with fewer side effects, but they will not reinstate tolerance and are, thus, not long-lasting or curative. Antigen-specific immunotherapies aiming to restore tolerance to the autoantigen under attack by specifically targeting only the part of the immune system that has gone awry while leaving the rest intact are, therefore, considered the holy-grail for treatment of autoimmune diseases.
- Antigen-specific tolerization approaches based on the a1-ECD have been studied earlier, using the oral or intranasal but not the intravenous route of administration. Delivery via the intravenous route could exploit a natural noninflammatory path, readily perfusing several organs with resident immune cells which have developed unique mechanisms for induction and maintenance of tolerance.
- T cell epitope peptides Another reasoning for using T cell epitope peptides is to avoid administration of antigens bearing conformational epitopes that may be recognized by B cells.
- Studies in the rat EAMG model have shown that oral administration of an a1-ECD construct with more native conformation resulted in disease exacerbation, while treatment with a similar but less native fragment was able to suppress ongoing EAMG (J Immunol (Baltimore Md: 1950) (2000) 165: 3599-3605).
- J Immunol (Baltimore Md: 1950) (2000) 165: 3599-3605) J Immunol (Baltimore Md: 1950) (2000) 165: 3599-3605).
- our results show that antigen conformation did not negatively affect the efficiency of treatment, since the P. pastoris protein used in most of the studies herein has a near native conformation, perhaps owing to differences in mechanism of action between the two administration routes.
- Protein glycosylation has been found to be important in regulating immune responses, and different glycans can lead to proinflammatory or immunosuppressive signals, although the mechanisms are far from being fully understood ( Front Immunol (2016) 9: 2754). Glycosylation of antigen used for therapy has been reported to play a role in i.v. tolerance induction in the case of EAE (Exp Neurol (2015) 267: 254-267). In our model of i.v. tolerance, however, protein glycosylation did not appear to play a major role, since there was no difference in the therapeutic efficacy between the P. pastoris and the E. coli expressed proteins. This was highly surprising.
- this example describes an effective treatment of disease in the EAMG model by intravenous delivery of a recombinant soluble major MG autoantigen, a1 -ECD m .
- a1 -ECD m represents a promising and efficient therapeutic approach for antigen-specific treatment.
- a1- ECDm shows a dose dependent capacity to induce remission after a short two-week treatment regimen.
- it provides an alternative route for clinical translation of antigen-based treatment of autoimmune diseases, as the makeup of the recombinant protein, comprising multiple auto-epitopes present in their native context, reduces the need for personalized tailoring of peptide-based treatments to overcome interindividual variability.
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WO1994000148A1 (en) | 1992-06-18 | 1994-01-06 | Yeda Research And Development Co. Ltd. | Synthetic peptides for the treatment of myasthenia gravis |
WO1996040777A2 (en) | 1995-06-07 | 1996-12-19 | Yeda Research And Development Co. Ltd. | Synthetic peptides and pharmaceutical compositions comprising them |
WO1998050544A1 (en) | 1997-05-07 | 1998-11-12 | Yeda Research And Development Co. Ltd. | Recombinant fragments of the human acetylcholine receptor and their use for treatment of myasthenia gravis |
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WO1994000148A1 (en) | 1992-06-18 | 1994-01-06 | Yeda Research And Development Co. Ltd. | Synthetic peptides for the treatment of myasthenia gravis |
WO1996040777A2 (en) | 1995-06-07 | 1996-12-19 | Yeda Research And Development Co. Ltd. | Synthetic peptides and pharmaceutical compositions comprising them |
WO1998050544A1 (en) | 1997-05-07 | 1998-11-12 | Yeda Research And Development Co. Ltd. | Recombinant fragments of the human acetylcholine receptor and their use for treatment of myasthenia gravis |
Non-Patent Citations (12)
Title |
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ANN NEW YORK ACAD SCI, vol. 778, 1996, pages 258 - 272 |
EXP NEUROL, vol. 267, 2015, pages 254 - 267 |
FRONT IMMUNOL, vol. 9, 2018, pages 2754 |
INT J BIOL MACROMOL, vol. 63, 2014, pages 210 - 217 |
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 63, 2014, pages 210 - 217 |
J IMMUNOL, vol. 165, 2000, pages 3599 - 3605 |
J NEUROIMMUNOL, vol. 278, 2015, pages 19 - 25 |
J NEUROIMMUNOL, vol. 303, 2017, pages 13 - 21 |
MACROMOL, vol. 63, 2014, pages 210 - 217 |
MOL PHARMACEUT, vol. 11, 2014, pages 1591 - 1598 |
MULT SCLER, vol. 5, no. 1, 1999, pages 2 - 9 |
MUSCLE NERVE, vol. 13, 1990, pages 485 - 492 |
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